The overall goal of this proposal is to develop a formulated combination microbicide which will prevent the spread of human immunodeficiency virus (HIV) both vaginally and rectally utilizing multiple protective factors which inactivate the virus at different stages in its replication cycle. Inhibition of HIV attachment to the CD4 cellular receptor will be accomplished by formulating plant-derived flavonoids with sulfated polysaccharides (carrageenans). There will therefore be redundancy built into the microbicide to inhibit HIV binding to its cellular receptor. Virucidal compounds, which destroy the viral envelope, will also provide redundant protection from infection. Both the antiviral ether lipid 1-0-octylsn- glycerol and citric acid will destabilize the envelopes of viral particles. Furthermore, the HIV reverse transcriptase will be inactivated by both antiviral flavonoids and a non-nucleoside reverse transcriptase inhibitor (Dr. Parniak, Project 1). Herpes simplex virus (HSV) will also be targeted by flavonoids, carrageenans, 1-0-octyl-sn-glycerol and citric acid to reduce genital ulceration and consequently the transmission of HIV to a greater extent than inactivating HIV only. Methods will be developed to quantify antiviral agents at each step in the pre-formulation and formulation process and physical and chemical pre-formulation data including solubility, stability, partitioning, and permeability data will be developed as part of these studies. Once active agents have been selected, their compatibility and toxicity with normal vaginal microflora and local tissues in both the isolated and formulated state will be determined. Following the initial formulation and development of a combination microbicide product, the microbicide will be optimized to maximize each antiviral mechanism and minimize toxicity in an iterative manner. The final formulated product will undergo stability testing, and product assessments will be made to ensure that the product has appropriate physical, chemical, microbiological, and antiviral properties during its shelf life. This project contributes to the program by producing new combinations of formulated microbicides based upon inhibition of viral replication using multiple and redundant antiviral mechanisms. Formulated combination microbicides produced in this project will be evaluated in vitro against HIV (Dr. Parniak, Project 1; Dr. Gupta, Project 2) and normal vaginal flora (Microbiology Core, Dr. Hillier), and as well as in humans (Dr. Landers, Project 4).